This project has been aimed at understanding the mechanism of dentin and bone mineralization, with specific attention to the roles of the organic matrix components in regulating the location and nucleation of mineral deposition, and defining the sizes and shapes of the resultant mineral crystals. We have, over the years, isolated the dentin proteins, particularly the acidic phosphorylated proteins (PP) biochemically, studied their properties, structures and tissue distribution, cloned some of them and prepared them as recombinant proteins. These studies have led us to two basic hypotheses: 1) The acidic PP have a dual role, localizing nucleation by binding specifically to the collagen fibrils and binding to preferential faces of the growing crystals to control crystal growth. 2) The same general schema is followed in all matrix mediated biomineralization processes, including those in invertebrate systems. Five specific aims are proposed, directed at validating (or not) these hypotheses. Aim 1 is to study the biosynthetic processing of the precursor, DSPP, to dentin sialoprotein (DSP) and PP. Organ culture and in vivo studies will be used. The relative fates of the DSP and PP portions will be determined. Aim 2 is to study the interactions of native rat incisor PP (RIPP) and rDMP2 with soluble monomeric type I collagen. Electron microscopy, dynamic and Rayleigh light scattering, and analytical ultracentrifugation will be used, along with molecular modeling approaches. Aim 3 will focus on the interactions of native RIPP, rDMP2 and other mineralized matrix NCPs with fibrillar type I collagen. The PP binding sites will be examined by TEM and biochemical means after in situ cross-linking. Aims 4 and 5 deal with the invertebrate system of sea urchin tooth mineralization. In Aim 4 the mineral bound tooth proteins will be purified, characterized and N-terminal sequenced. A cDNA library constructed from urchin tooth mRNA has been created and will be probed for the mineral phase related proteins by screening with anti-DMP2, and other antibodies. Selected cDNAs will be cloned and their complete sequences determined. Recombinant proteins will be prepared and studied. In Aim 5, high resolution microCT scanning and other x-ray techniques will be used to correlate mineral and protein distributions. These data should provide a clearer understanding of the mechanisms whereby the acidic matrix proteins nucleate and control mineral deposition, and link the vertebrate and invertebrate systems.